Combining CRISPR activation and interference capabilities using dCas9 and G-quadruplex structures

We demonstrate that both Clustered regularly interspaced short palindromic repeats (CRISPR) interference and CRISPR activation can be achieved at RNA and protein levels by targeting the vicinity of a putative G-quadruplex (GQ)-forming sequence (PQS) in the c-Myc promoter with nuclease-dead Cas9 (dCas9). The achieved suppression and activation in Burkitt's Lymphoma cell line and in in vitro studies are at or beyond those reported with alternative approaches. When the template strand (contains the PQS) was targeted with CRISPR-dCas9, the GQ was destabilized and c-Myc mRNA and protein levels increased by 2.1- and 1.6-fold, respectively, compared to controls in the absence of CRISPR-dCas9. Targeting individual sites in the nontemplate strand (NTS) with CRISPR-dCas9 reduced both the c-Myc mRNA and protein levels (by 1.8- and 2.5-fold, respectively), while targeting two sites simultaneously further suppressed both the mRNA (by 3.6-fold) and protein (by 9.8-fold) levels. These were consistent with cell viability assays when single or dual sites in the NTS were targeted (1.7- and 4.7-fold reduction in viability, respectively). We also report extensive in vitro biophysical studies which are in quantitative agreement with these cellular studies and provide important mechanistic details about how the transcription is modulated via the interactions of RNA polymerase, CRISPR-dCas9, and the GQ.